SUMMARY Alcohol use disorders are in part characterized by the loss of control over alcohol drinking and the emergence of negative emotionality during abstinence from alcohol. While the brain regions and neurotransmitter systems affected by chronic alcohol exposure are well characterized, our understanding of the specific neural circuits driving drinking escalation and negative affect during withdrawal remains limited. We have generated data indicating that neurons located in the parasubthalamic nucleus (PSTN), a small region of the posterior lateral hypothalamus whose function in addiction is currently unknown, may be a critical component of this circuitry. Specifically, we show that chemogenetic stimulation of PSTN neurons expressing the neuropeptide corticotropin- releasing factor (CRF) replicates the behavioral symptomatology of withdrawal and that PSTN neurons become activated in ethanol-dependent mice experiencing withdrawal. A first aim of this proposal is to explore the mechanisms underlying the phenotypes resulting from the chemogenetic activation of PSTN CRF neurons. We will evaluate the contribution of PSTN neurons projecting to the central nucleus of the amygdala (as opposed to other targets of the PSTN) and the role of CRF signaling (as opposed to other neurotransmitters co-released by PSTN CRF neurons). Another goal is to test the hypothesis that the PSTN is a critical node of the neuronal network driving the behavioral symptomatology of ethanol withdrawal. We will determine whether the activation of PSTN neurons is necessary to the expression of withdrawal symptoms in dependent mice, whether it drives the activation of downstream central amygdala neurons, and which neurotransmitter is implicated. Finally, we aim to understand how PSTN neurons become activated during ethanol withdrawal. We will use retrograde tracing combined with c-Fos mapping as well as electrophysiological recordings to investigate the mechanisms controlling the activity of PSTN neurons in the context of ethanol withdrawal. Throughout the project, we will leverage state-of-the-art genetic tools for the functional manipulation of specific neural pathways, local silencing of gene expression and neuronal mapping, along with whole-brain imaging. In addition, we will use a well- validated mouse model of ethanol dependence that we have refined with novel measures of affective perturbation. Altogether, the proposed experiments are designed to enhance our understanding of the neural circuitry that contributes to motivational and emotional dysfunction in alcohol use disorders and may provide novel avenues for the development of more efficacious treatments.